The notochord plays a fundamental role in supporting and patterning the developing body plan in all chordate embryos. The transcription factor Brachyury is required for notochord formation and it is known to activate the transcription of numerous notochord genes in chordate embryos as different as sea squirts and humans. Mutations in the Brachyury coding region have been associated with vertebral malformations and increased risk of spina bifida. In addition, recent studies have indicated a direct association between the duplication of the human Brachyury locus and familial chordoma, a malignant notochord-derived tumor. The knowledge of the Brachyury-downstream gene cascade and, in particular, the identities of the transcription factors that act downstream of or in concert with Brachyury is still fragmentary. We use the basal chordate Ciona intestinalis (sea squirt), a simple yet informative vertebrate relative, as an accessible, fast-developing model system for identifying evolutionarily conserved transcriptional activators and for elucidating their hierarchical relationship with Ciona Brachyury (Ci-Bra). Our preliminary data, gathered from the comparative analysis of the structure and function of notochord cis-regulatory modules (CRMs) and from specific searches for notochord transcription factors evolutionarily conserved from Ciona to vertebrates, lead us to hypothesize that: 1) Ci-Bra controls part of its targets indirectly, via transcriptional intermediaries and 2) Ci-Bra controls part of its targets by acting synergistically with other transcription factors. We propose to characterize late-onset notochord transcription factors recently identified in our lab. Our main goals are to elucidate the role played by these factors in notochord development and to shed light on their hierarchical position within the Ci-Bra gene regulatory network. Our specific aims are: 1) Analyze the developmental role and targets of a newly identified transcription factor, and its relationship with Ci-Bra; 2) Identify the activators controlling the notochord CRMs identified in our lab and study their function in notochord development; 3) Characterize the role of Ci-Bra in late notochord differentiation through the analysis of one of its potential transcriptional intermediaries, Ci-XBP-1, and of its downstream targets. The broad scope of the proposed research is to exploit the experimental advantages of Ciona to rapidly gain basic insights into the mechanisms controlling gene expression in the notochord during development and evolution. In a wider perspective, this research aims to shed light on the molecular mechanisms employed by Brachyury to control its target genes, which, when compromised, are responsible for notochord malformations and notochord-derived tumorigenesis. Given the pervasive role of Brachyury in notochord formation in all chordates analyzed, it is likely that these findings will be applicable to vertebrates, including humans.